The Shiga toxin family consists of Shiga toxin, produced by Shigella dysenteriae 1, and the Shiga-like toxins (SLTs), produced by Escherichia coli. Only the production of moderate to high levels of Shiga toxin and SLT in vitro has been implicated in pathogenesis. All Shiga toxin molecules are composed of an enzymatic (A) subunit and multiple copies of a binding (B) subunit. The long term goal of this proposal is to investigate the regulation, assembly, and export of Shiga toxin. Four specific aims are proposed to achieve this goal: 1) The amount of Shiga toxin A and B subunits produced by S. dysenteriae will be measured and compared to the 5B:IA stoichiometry of the holotoxin. The effect that inducing overproduction of the binding subunit will be assessed using a B subunit gene (stxB) clone. Competitive binding assays will be used to investigate the hypothesis that free B subunits affect toxin levels in vivo by competing with the holotoxin for binding sites. 2) The following models for the transcriptional regulation of the stxA and stxB genes will be investigated using Northern blot, primer extension, and mutational analyses: i) a bicistronic mRNA with independent ribosome binding sites (rbs) for the stxA and stxB genes is transcribed from a promoter 5' to stxA; ii) the stxA and stxB genes are transcribed from separate promoters; or iii) endonucleolytic processing of a bicistronic mRNA which influences transcript stability may regulate toxin synthesis. 3) Site-directed mutagenesis and gene fusion will be used to investigate the hypothesis that the putative stxB gene rbs is responsible for overproduction of the B polypeptide. This model will be tested in vivo by assessing the virulence of E. coli strains expressing the mutated slt-II operon for mice. 4) The capacity of B subunits to form pentamers which may modulate virulence by competing with the holotoxin for receptor binding sites will be assessed using the stxB clone. The region of the A subunit which is required for holotoxin assembly will be established by constructing protein fusions. Ultimately, the A subunit residues which are required for holotoxin assembly will be identified using site-directed mutagenesis. This information will be used to investigate A subunit translocation domain(s).